In recent years the role of phosphoinositides in an important, novel system of signal transmission across cell membranes, has come to be recognized. In response to a stimulus, such as the binding of appropriate agonists to certain classes of receptors, phosphatidylinositol-4,5-bisphosphate (PIP2) is enzymatically cleaved to give rise to two second messenger molecules, inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) which can mobilize intracellular Ca2+ from nonmitochondrial stores and activate calcium- and phospholipid-dependent protein kinase C respectively. In the proposed investigation certain features of this system will be examined in the DDT1-MF2 smooth muscle cell line. The surface of these cells contains alpha 1-adrenergic receptors which, when stimulated with norepinephrine (NE), cause the appropriate response. It is likely that specific pools of PIP2, possibly with characteristic fatty acyl groups, presumably in the plasma membrane, are subject to the stimulated hydrolysis followed by regeneration through phosphatidic acid, phosphatidylinositol (PI) and PI-4-phosphate. In order to identify phospholipid and second messenger pools, cells will be incubated, under a variety of selected conditions which will yield specific radioactivities for kinetic analyses. Direct or chase incubations with time will be done for non-equilibrium kinetics and direct incubations at one time point for equilibrium kinetics, all with fixed or varying concentrations of modifying agents as appropriate. Other variables will be 1) NE or no agonist or NE+ antagonist and 2) 32Pi, (2-3H)inositol, (14C)-glycerol or (14C) fatty acid (arachidonic or oleic) as precursor. The strategy will be to compare unmodified series of incubations with those that have been altered in one of four ways: 1) interfering with PI synthesis through use of an analog of phosphatidyl-CMP; 2) depleting inositol through treatment of the cells with inositol dehydrogenase or oxidase; 3) inhibiting DAG phosphorylation with a DAG kinase inhibitor; or 4) activating the inositol exchange reaction with Mn2+. Separations will be by TLC for classes and by HPLC for molecular species of phospholipids; IP3, its isomers and other IPs will be isolated by column chromatography or HPLC. These investigations of a fundamental physiological phenomenon are relevant for diseases where Ca2+-mobilizing receptors or processes following from their activation such as secretion, growth, contraction, fertilization or sensory transduction may be affected. They have therefore broad health-related relevance.